WO2018170621A1 - Vector for reducing mir-148a and mir-424 expressions and application thereof - Google Patents

Abstract

Provided are a vector for reducing miR-148a and miR-424 expressions and application thereof. A construction method for the expression vector comprises ligating a Tud RNA targeting to miR148a and miR-424 to a pLKO.1-puro cloning vector to obtain a dual-miRNA inhibition expression vector pLKO-Tud-148a-424. The expression vector has the function of inhibiting the activity of has-miR-148a and has-miR-424.

Description

 Title of Invention: Vectors for Reducing Expression of miR-148a and miR-424 and Their Applications

 [0001] The present invention relates to the field of molecular biology, and more particularly to a vector for reducing the expression of miR-148a and miR-424 and uses thereof.

 Background technique

 [0002] RNA is an important substance in living organisms and plays various functions in life activities. RNA can be classified into messenger RNA (mRNA) and non-coding RNA depending on whether the protein is encoded or not.

(non-coding RNA, ncRNA). Small RNA (small

 RNA, smRNA) is an important class of ncRNAs. miRNAs are endogenous small RNAs in organisms that are typically 20-24 nt in length. The miRNA is part of the pri-miRNA (primary RNA) and is initially expressed in the nucleus by RNA polymerase Π. The mature miRNA acts as a guiding molecule, binds to the target gene mRNA according to the principle of base pairing, and directs the silencing complex (RISC) to degrade mRNA or hinder its translation, thereby exerting a negative regulation effect on the expression of the target gene.

 [0003] miR-148a is a microRNA that has been studied more in recent years. It has been reported that miR-148a is closely related to exogenous substance metabolism, apoptosis, occurrence, development and epigenetics of various cancers, so it is important to study the function of miR-148a; miR-424 is in recent years A miRNA discovered, which acts on a target gene in a variety of tumors and participates in a signal pathway of target gene regulation, thereby affecting the biological effects and development of tumor cells, exerting effects similar to oncogenes, tumor suppressor genes, or promoting Inhibit the invasion and metastasis of tumors. Studies have shown that miR-424 is a multifunctional miRNA, which is associated with cell invasion and metastasis of cervical cancer, pancreatic cancer, etc.; it is associated with the expression of inflammatory factors such as IL-6 and TNF-ot; since the miR-424 promoter region has CpG islands It is also associated with methylation-induced gene silencing. By controlling the expression of miR-148a and miR-424, the synergy with other drugs can provide new epigenetic ideas for the treatment of cancer. technical problem

 [0004] MiRNA functional studies often require miRNA silencing techniques, including anti-miR, antago miR, miRNA

Sponge, etc. These technologies are all transient transfection techniques and cannot maintain long-term stability of the target miRNA. Silence, the effect of silence is far from optimal.

[0005] Tough Decoy RNA (Tud RNA) is a novel miRNA-inhibiting miRNA that inhibits miRNA by introducing double-stranded RNA to target miRNAs. Because the inserted RNA is double-stranded and has a secondary structure of stem-loops, it is resistant to intracellular nuclease degradation and inhibits miRNAs in a long-term, stable, and efficient manner.

 Problem solution

 Technical solution

 The technical problem to be solved by the present invention is to provide a dual miRNA suppression expression vector which is simple in structure, low in cost and simple in operation.

 [0007] A Tud RNA targeting a dual miRNA, the nucleotide sequence of which is shown in SEQ ID NO: 1 in the Sequence Listing.

A dual miRNA-inhibiting expression vector comprising the sequence of the invention SEQ ID NO: 1.

[0009] The use of the dual miRNA-inhibiting expression vector of the present invention in tumor therapeutic research, the double miRNA-inhibiting expression vector can inhibit the expression of miR-148a and miR-424 after transformation of Hela cells.

[0010] A method for constructing a dual miRNA suppression expression vector according to the present invention comprises the following steps:

(1) Designing a dual miRNA-targeted Tud RNA Seq1 according to the miR-148a and miR-424 sequences and the principle of Tud RNA technology, the nucleosides of the miR-148a and miR-424 sequences The acid sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3 in the Sequence Listing, respectively, and the nucleotide sequence of the Seq1 sequence is shown in SEQ ID NO: 1 of the Sequence Listing, and was commissioned by Shanghai Biotech for synthesis.

(2) The synthesized sequence is two complementary single-stranded DNAs. The two single-stranded DNAs were dissolved in ddH20, mixed in an equimolar ratio, treated at 95 ° C for 5 min, and then allowed to cool to room temperature at room temperature.

[0013] (3) extraction vector pLK0.1-puro, double digestion with Age I and Eco RI enzyme 1

 After h, the digested vector was recovered using the MinElute Reaction Cleanup Kit, and the TuD obtained in the previous step was further treated with T4 DNA ligase.

The RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-TuD-148a-424, and finally the ligation product was transformed into competent E. coli Stbl3 and plated onto a plate containing ampicillin LB medium. Incubate at 37 °C for 14 h. Single colonies were picked and sequenced. The correct bacteria were amplified and cultured and extracted with a small amount of endotoxin-free extraction kit. The extracted plasmid is the homologous interference pLKO- required for the invention. Plasmid for TuD-148a-424.

 Advantageous effects of the invention

 Beneficial effect

 [0014] The homologous interference miR-148a and miR-424 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA has higher binding efficiency than the currently used single-stranded miRNA sponge. And the same target for two targets, can better achieve the interference of two miRNAs, improve the efficiency of miRNA function research.

 Brief description of the drawing

 DRAWINGS

 Figure 1 shows the miRNA expression levels of 16HBE cells and TuD-148a-424 cells, wherein a.

 Expression of miR-148a, b. expression of miR-424.

 BEST MODE FOR CARRYING OUT THE INVENTION

 BEST MODE FOR CARRYING OUT THE INVENTION

[0016] The present invention can be better understood in light of the following examples. However, those skilled in the art will readily understand

The specific material ratios, process conditions, and results of the examples are merely illustrative of the invention and should not be construed as limiting the invention as described in the claims.

The lentiviral plasmid pLKO.l-puro vector used in the present invention was purchased from Addgene; the human bronchial epithelial cells (16HBE cell strain) used in the present invention were purchased from ATCC, USA.

[0018] Example 1 Design and Synthesis of TuD RNA Targeting miR-148a and miR-424

[0019] Based on the TuD RNA design sequence and the sequence information of miR-148a and miR-424 provided in miRBase, the TuD RNA oligonucleotide sequence targeting miR-148a and miR-424 was designed, and its sequence is SEQ ID.

ΝΟ:1, commissioned by Shanghai Biotech to synthesize by means of gene synthesis.

[0020] Example 2 annealing of the sequence

 [0021] The synthesized sequence is two complementary single-stranded DNAs. The two single-stranded DNAs were dissolved in ddH20, mixed at an equimolar ratio, treated at 95 ° C for 5 min, and allowed to cool to room temperature by allowing them to stand at room temperature.

[0022] Example 3 Recombinant Construction of pLKO-Tud-148a-424 Lentiviral Recombinant Vector

[0023] The vector pLK0.1-puro was extracted and double-digested with Age I and Eco RI for 16 h, using MinElute Reaction Cleanup Kit recovers the digested vector and uses T4 DNA

 Ligase will get TuD from the previous step

 The RNA sequence was ligated into the vector pLKO.l-puro to form the recombinant vector pLKO-Tud-148a-424, and finally the ligation product was transformed into competent E. coli Stbl3 and plated onto a plate containing ampicillin LB medium. Incubate at 37 °C for 14 h. Single colonies were picked and sequenced. The correct sequencing bacteria were expanded and extracted with a non-endotoxin plasmid miniprep kit. The extracted plasmid was the plasmid for the homologous interference pLKO-TuD-148a-424 required by the present invention.

Example 4 Plasmid of pLKO-TuD-148a-424 16HBE cell

 [0025] 16HBE cells were seeded in 6-well plates, 1000000 cells per well, and the cell density was about 60% after 18 hours. The plasmid pLKO-TuD-148a-424 was transduced into 16HBE cells with Lipfectamine 2000, and culture was continued for 48 h. Thereafter, the cells were cultured for 3 days in DMEM medium containing 1.0 g/ml puromycin, and the cell line obtained by the screening was named TuD-148a-424 cell line.

 Example 5 Fluorescence Quantitative PCR Detection of Changes in Expression Levels of miRNAs

 [0027] Normal 16HBE cells and TuD-148a-424 cells were inoculated into 6-well plates, respectively, and the cells were cultured for about 24 hours to a degree of fusion of 80%. The miRNAs of these cells were extracted using the miRcute miRNA extraction and isolation kit, and the corresponding cDNA was obtained by reverse transcription. Take 2 kinds of cells of cDNA 2

 As a template, real-time PCR was used to detect the expression levels of miR-148a and miR-424, respectively. The experiment was repeated three times, and three parallel samples were set per well to snord.

 44 as an internal reference. The results are shown in Figure 1. It can be seen that the expression level of miR-148a in TuD-148a-424 cells is 52% lower than that in 16HBE cells, and the expression level of miR-424 is 62% lower than that in 16HBE cells. The difference is statistically significant ( p<0.01), indicating that the TuD-148a-424 cell line was successfully constructed.

 Industrial applicability

 [0028] The homologous interference miR-148a and miR-424 TuD RNA sequences designed by the present invention have a stem-loop structure and are not easily degraded, and the double-stranded Tud RNA has higher binding efficiency than the currently used single-stranded miRNA sponge. And the same target for two targets, can better achieve the interference of two miRNAs, improve the efficiency of miRNA function research.

Claims

Claim  [Claim 1] A Tud RNA targeting a dual miRNA, characterized in that the nucleotide sequence thereof is shown in SEQ ID NO: 1 of the Sequence Listing.  [Claim 2] A vector for reducing expression of miR-148a and miR-424, characterized in that it comprises the sequence of claim 1.  [Claim 3] The use of the dual miRNA-inhibiting expression vector of claim 2 for tumor therapeutic research, wherein the dual miRNA-inhibiting expression vector inhibits the expression of human miR-148a and miR-424.  [Claim 4] A method for constructing a dual miRNA-inhibiting expression vector according to claim 2, comprising the steps of:  (1) Designing a dual miRNA-targeted Tud RNA Seq1 according to the miR-148a and miR-424 sequences and the principle of Tud RNA technology, wherein the nucleotide sequences of the miR-148a and miR-424 sequences are respectively As shown in SEQ ID NO: 2 and SEQ ID NO: 3 in the Sequence Listing, the nucleotide sequence of the Seq1 sequence is shown in SEQ ID NO: 1 of the Sequence Listing, and was commissioned by Shanghai Biotech for synthesis. (2) The synthesized sequence is two complementary single-stranded DNAs. The two single-stranded DNAs were dissolved in ddH ₂ 0, mixed in an equimolar ratio, treated at 95 ° C for 5 min, and allowed to stand at room temperature to allow them to naturally cool to room temperature.  (3) The extraction vector pLK0.1-puro, treated with Age I and Eco RI enzymes for 1 h The recombinant vector was ligated with the T4 DNA ligase, and the TuD RNA sequence obtained in the previous step was ligated into the vector pLKO.l-puro to form a recombinant vector pLKO-TuD-148a-424, and finally the ligated product was transformed into a sensation. The cells were plated in E. coli Stbl3 and plated on ampicillin-containing LB medium and cultured at 37 °C for 14 h. Pick a single colony and sequence it. The correct sequencing bacteria were expanded and extracted with an endotoxin-free plasmid extraction kit. The extracted plasmid was the plasmid of the same interference interference pLKO-TuD-148a-424 required by the present invention.